Nucleic acid-based genetic methods for identification of microorganisms have greatly reduced the time and labor involved in clinical diagnosis. Such methods include, for example, nucleic acid hybridization (e.g., Southerns/microarrays and slot blots), nucleotide sequencing, nucleic acid cloning techniques, restriction digestion of nucleic acids and nucleic acid amplification. In particular, nucleic acid amplification has provided means for rapid, sensitive and specific identification of microorganisms by amplification and detection of specific genes or gene fragments. For use as diagnostic methods, it is of particular interest to apply these nucleic acid analyses to biological samples such as plasma and whole blood samples. Prior to the availability of nucleic acid-based methods for detection and identification of microorganisms, plasma or blood samples were analyzed for the presence of microorganisms by blood culturing. However, processing of clinical samples for nucleic acid analyses requires different criteria than sample processing for culturing. For example, nucleic acids must be released from the microorganism in a form suitable for the analysis; nucleic acids must be present in a composition with the appropriate components, ionic strength and pH for the biochemical reactions of the analysis; and inhibitors of the reactions such as nucleases, if present in the clinical sample or introduced during sample processing, must be removed or rendered non-inhibitory.
One potential biochemical detection method involves the use of nucleic acid hybridization. The sequence specificity embodied in nucleic acids makes it possible to differentiate virtually any two species by nucleic acid hybridization. Standard techniques for detection of specific nucleotide sequences generally employ nucleic acids that have been purified away from cellular proteins and other cellular contaminants. The most common method of purification involves lysing the cells with sodium dodecyl sulfate (SDS), digesting with proteinase K (ProK), and removing residual proteins and other molecules by extracting with organic solvents such as phenol, chloroform, and isoamylalcohol.
Endogenous nucleases released during cell solubilization can frustrate efforts to recover intact nucleic acids, particularly ribonucleic acids (RNA). While deoxyribonucleases (DNases) are easily inactivated by the addition of chelating agents to the lysis solution, ribonucleases (RNases) are far more difficult to eliminate. RNases are ubiquitous, being present even in the oil found on human hands. Accordingly, protecting against RNase is a commonly acknowledged aspect of any standard RNA preparation technique. The standard procedure for preparing laboratory stocks of pancreatic RNase is to boil a solution of the enzyme for 15 minutes. The purpose of this treatment is to destroy all traces of contaminating enzyme activity because other enzymes cannot survive boiling.
Sambrook, et al., Molecular Cloning, 3rd Edition (2001), a compendium of commonly followed laboratory practices, recommends extensive precautions to avoid RNase contamination in laboratories. Such precautions include preparing all solutions that will contact RNA using RNase-free glassware, autoclaved water, and chemicals reserved for work with RNA that are dispensed exclusively with baked spatulas. Besides purging laboratory reagents of RNase, RNase inhibitors are typically included in lysis solutions. These are intended to destroy endogenous RNases that generally become activated during cell lysis. Also, it is common practice to solubilize RNA in diethyl pyrocarbonate (DEPC)-treated water. Moreover, in an attempt to improve the handling of RNA samples, formamide has been tested as a solubilizing agent for the long-term storage of RNA. Chomczynski, P., Nucleic Acids Research 20, 3791-3792 (1992).
Protecting against RNase is cumbersome and costly, and typical extraction procedures require the handling of caustic solvents, access to water baths, fume hoods, and centrifuges, and even the storage and disposal of hazardous wastes. The direct analysis of unfractionated solubilized biological samples would avoid the cost and inconvenience of these purification techniques.
In view of the foregoing, there exists a need for a simple and rapid method by which biological samples such as plasma and blood may be treated for the extraction therefrom of nucleic acid for analysis.